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DNA-only Transposons

DNA-only Transposons

DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
The donor site from where the transposon is excised is either degraded or...

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Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

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An integrative expression vector for Actinosynnema pretiosum.

Shan Goh¹, Andrea Camattari, Daniel Ng

¹Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore. Shan.Goh@ki.se

BMC Biotechnology

|October 25, 2007

Summary

Researchers developed new integrative vectors for Actinosynnema pretiosum, enabling constitutive gene expression. These tools facilitate ansamitocin P-3 (AP-3) biosynthesis studies without impacting AP-3 production or cell growth.

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Area of Science:

Microbiology
Molecular Biology
Biotechnology

Background:

Actinosynnema pretiosum ssp. auranticum produces ansamitocin P-3 (AP-3), a valuable antitumor agent.
Enhancing AP-3 production requires overcoming biosynthetic pathway bottlenecks through gene expression.
A lack of suitable integrative expression vectors for A. pretiosum hinders such efforts.

Purpose of the Study:

To construct a novel integrative expression vector for heterologous gene expression in A. pretiosum.
To enable constitutive gene expression for metabolic engineering of AP-3 biosynthesis.
To provide a tool for fundamental research into AP-3 production pathways.

Main Methods:

Construction of integrative expression vectors incorporating IS117 transposase, ermE* promoter, and various ribosome-binding sites (RBS).
Use of xylE as a translational reporter gene for easy assessment of gene expression via catechol assay.
Transformation of E. coli and generation of A. pretiosum transconjugants to test vector functionality.

Main Results:

Developed several integrative vectors, with pAP42 (E. coli consensus RBS) and pAP43 (asm19 RBS) showing strong and moderate expression, respectively.
Created an operon construct for multi-gene expression, demonstrating vector versatility.
Identified four distinct plasmid integration sites in transconjugants, with no adverse effects on AP-3 levels or cell growth.

Conclusions:

A suite of integrative vectors for constitutive gene expression in A. pretiosum has been successfully developed.
The vectors allow for straightforward, colorimetric assessment of gene translation on agar plates.
These vectors are well-suited for investigating AP-3 biosynthesis without negatively impacting AP-3 yields.